Cytokines and hormones are secreted proteins that bind to cell surface receptors and activate cellular differentiation and proliferation through a cascade of intracellular signaling events. They include insulin, erythropoietin (EPO), granulocyte colony stimulating factor (GCS F), thrombopoietin (TPO), human growth hormone (hGH), vascular endothelial growth factor (VEGF), angiostatin, endostatin, insulin, the interleukins, and the interferons. In general, each cytokine has a specific cell-surface receptor. These receptors comprise three major domains: an extracellular portion that binds the cytokine, a transmembrane domain to anchor the receptor in the membrane, and an intracellular signaling domain that is activated by cytokine binding.
Cytokines generally have at least two binding sites, and sometimes three, for the receptors. Monomeric receptors are brought together by cytokine binding, to result in the formation of a receptor oligomer. This oligomer is the biologically active form and is necessary for a variety of intracellular receptor signaling events.
From a commercial perspective, cytokines are used to treat millions of patients for anemia, cancer, diabetes, neurological and growth disorders. However, cytokines are generally large molecules that must be administered by intravenous or subcutaneous injection. Accordingly, the pharmaceutical industry has been highly motivated to develop small molecule replacement that can be taken orally. Thus, there is enormous commercial interest in finding cytokine-mimetic drugs that could eliminate the need for injection and lower the cost of producing the recombinant proteins.
However, a variety of technical barriers have prevented the discovery and commercialization of small molecule mimics for cytokines. The development of small molecule cytokine mimics is blocked by the difficulty in reconstituting the biologically relevant receptor structure, a precisely oriented receptor oligomer. So far it has not been possible to reconstitute the active receptor oligomer for use in in vitro drug screening assays, aiming to isolate cytokine mimetics. Current screening approaches utilize receptor molecules in random orientations, not as functional dimers or trimers, thereby screening for receptor affinity rather than activity. Cell-based assays have recently been developed that present receptors in a ‘natural’ manner however, these assays are difficult to use and limited in screening power for high throughput screening.
De novo protein design has received considerable attention recently, and significant advances have been made toward the goal of producing stable, well-folded proteins with novel sequences. Efforts to design proteins rely on knowledge of the physical properties that determine protein structure, such as the patterns of hydrophobic and hydrophilic residues in the sequence, salt bridges and hydrogen bonds, and secondary structural preferences of amino acids. Various approaches to apply these principles have been attempted. For example, the construction of α-helical and β-sheet proteins with native-like sequences was attempted by individually selecting the residue required at every position in the target fold (Hecht et al., Science 249:884-891 (1990); Quinn et al., Proc. Natl. Acad. Sci USA 91:8747-8751 (1994)). Alternatively, a minimalist approach was used to design helical proteins, where the simplest possible sequence believed to be consistent with the folded structure was generated (Regan et al., Science 241:976-978 (1988); DeGrado et al., Science 243:622-628 (1989); Handel et al., Science 261:879-885 (1993)), with varying degrees of success. An experimental method that relies on the hydrophobic and polar (HP) pattern of a sequence was developed where a library of sequences with the correct pattern for a four helix bundle was generated by random mutagenesis (Kamtekar et al., Science 262:16801685 (1993)). Among non de novo approaches, domains of naturally occurring proteins have been modified or coupled together to achieve a desired tertiary organization (Pessi et al., Nature 362:367-369 (1993); Pomerantz et al., Science 267:93-96 (1995)).
Though the correct secondary structure and overall tertiary organization seem to have been attained by several of the above techniques, many designed proteins appear to lack the structural specificity of native proteins. The complementary geometric arrangement of amino acids in the folded protein is the root of this specificity and is encoded in the sequence.
Several groups have applied and experimentally tested systematic, quantitative methods to protein design with the goal of developing general design algorithms (Hellingia et al., J. Mol. Biol. 222: 763-785 (1991); Hurley et al., J. Mol. Biol. 224:1143-1154 (1992); Desjarlaisl et al., Protein Science 4:2006-2018 (1995); Harbury et al., Proc. Natl. Acad. Sci. USA 92:8408-8412 (1995); Klemba et al., Nat. Struc. Biol. 2:368-373 (1995); Nautiyal et al., Biochemistry 34:11645-11651 (1995); Betzo et al., Biochemistry 35:6955-6962 (1996); Dahiyat et al., Protein Science 5:895-903 (1996); Dahiyat et al., Science 278:82-87 (1997); Dahiyat et al., J. Mol. Biol. 273:789-96; Dahiyat et al., Protein Sci. 6:1333-1337 (1997); Jones, Protein Science 3:567-574 (1994); Konoi, et al., Proteins: Structure, Function and Genetics 19:244-255 (1994)). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration. In particular, WO98/47089, and U.S. Ser. No. 09/127,926 describe a system for protein design; both are expressly incorporated by reference.
A need still exists for a method of screening for cytokine mimetics. Thus, it is an object of the present invention to provide non-naturally occurring cell surface receptor analogs, capable of binding naturally occurring ligands, such as cytokines. It is a further aspect of this invention to provide nucleic acids encoding the receptor analogs and methods of using the receptor analogs for screening cytokine mimetics.